CA2956588A1 - High isomerohydrolase activity mutants of mammalian rpe65 - Google Patents

Abstract

Mutant mammalian RPE65 proteins and portions thereof, and nucleic acids encoding the mutants, for use in treating a condition related to retinal degeneration in a subject, the mutant mammalian RPE65 proteins or portions thereof having isomerohydrolase activity. A gene therapy method of treating a condition related to retinal degeneration in a mammalian subject in need of such treatment, comprising administering to the subject a therapeutically- effective amount of a vector comprising a nucleic acid encoding a mutant mammalian RPE65 protein or a portion thereof. A method of treating a condition related to retinal degeneration in a subject in need of such treatment, comprising administering to the subject a therapeutically-effective amount of at least one of a mutant mammalian RPE65 protein or a portion thereof.

Description

CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional application Serial No. 62/031,472, filed July 31, 2014, which application is expressly incorporated herein by reference in its entirety.
GOVERNMENT SUPPORT

[0002] This invention was supported by National Institutes of Health grants EY012231, EY018659, EY019309, and GM104934, a grant from IRRF. The government has certain rights in the invention.
INTRODUCTION

[0003] Retinal pigment epithelium (RPE) specific 65-kDa protein (RPE65) is essential for metabolism of vitamin A in the eye and for maintenance of normal vision. It is a key retinoid visual cycle enzyme that catalyzes isomerization of all-trans retinyl ester to 11-cis retinol (11 cROL), the precursor of chromophore of visual pigments. Mutatio ns of RPE65 are associated with inherited retinal dystrophies such as Leber's Congenital Amaurosis (LCA) and Retinitis Pigmentosa (RP). Previous RPE65 gene replacement therapy in RPE65 null-mutants of dog and mouse models displayed promising effects on retinal degeneration.
However, mammalian RPE65, such as human RPE65 (hRPE65), has lower specific activity than other retinoid processing enzymes, and high abundance of RPE65 in the RPE
(11 ug/eye in bovine) is thus required to generate sufficient 11-cis retinoid for normal vision. This demand for high levels of hRPE65 limits the efficacy of RPE65 gene therapy.

[0004] [0001]Several vectors for gene delivery, such as adenovirus (AD), recombinant adeno-associated virus (rAAV), lentivirus, plasmid incorporated in nanoparticles and plasmid DNA with electroporation have been used to deliver intact DNA (or reporter genes) to the ocular tissues. Gene delivery into the subretinal space using AD and lentiviral vectors expressing GFP showed widely distributed GFP expression. Recent human clinical trials using rAAV expressing wild type (wt) human RPE65 (rAAV-hRPE65) showed only modest improvements of vision in patients with RP and LCA. However, no prior RPE65 gene therapies have successfully generated full vision recovery in spite of successful gene delivery.

SUMMARY

[0005] The presently disclosed inventive concepts include, but are not limited to, mutant mammalian RPE65 proteins or portions thereof that include amino acid sequences having at least 90% identity with at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17;

having isomerohydrolase activity; and having an amino acid substitution in at least one of positions 170 and 297.

[0006] In certain non-limiting embodiments, the amino acid substitution in position 170 may be Lys, Arg, or His.

[0007] In certain non-limiting embodiments, the amino acid substitution in position 297 may be Gly or Ala.

[0008] In certain non-limiting embodiments, the mutant mammalian RPE65 protein or portion thereof may include one or more amino acid substitutions in positions 2, 3, and 26.

[0009] In certain non-limiting embodiments, the amino acid substitution in position 2 may be Tyr, Phe, or His.

[0010] In certain non-limiting embodiments, the amino acid substitution in position 3 may be Ser, Thr, or Cys

[0011] In certain non-limiting embodiments, the amino acid substitution in position 26 may be Val, Ala, or Ile.

[0012] In certain non-limiting embodiments, the mutant mammalian RPE65 protein or portion thereof may include amino acid substitutions in each of positions 2, 3, 26, 170, and 297, which may be, for example, those described herein.

[0013] In certain non-limiting embodiments, the mutant mammalian RPE65 protein or portion thereof has greater isomerohydrolase activity than non-mutated RPE65 protein.

[0014] In certain non-limiting embodiments, the mutant mammalian RPE65 protein or portion thereof has greater isomerohydrolase activity than human RPE65 having the amino acid sequence set forth in SEQ ID NO:1 or bovine RPE65 having the amino acid sequence set forth in SEQ ID NO:9.

[0015] Also disclosed herein are pharmaceutical compositions that include a mutant mammalian RPE65 protein or portion thereof described herein, disposed in a pharmaceutically-acceptable carrier or vehicle.

[0016] Also disclosed herein are nucleic acids that encode a mutant mammalian RPE65 protein or portion thereof described herein.

[0017] In certain non-limiting embodiments, the nucleic acid may be disposed within a vector.

[0018] In certain non-limiting embodiments, the vector includes a promoter or enhancer sequence operatively-linked to the nucleic acid; and/or includes a stop codon or a poly-A
sequence located 3' of the nucleic acid.

[0019] In certain non-limiting embodiments, the vector includes an RPE65 promoter sequence that is operatively-linked to the nucleic acid; and/or includes a CMV

enhancer/chicken f3-actin promoter sequence operatively-linked to the nucleic acid.

[0020] In certain non-limiting embodiments, the vector is selected from the group consisting of adenovirus, adeno-associated virus (AAV), nanoparticles, plasmids, and lentivirus.

[0021] Also disclosed herein are AAV vectors that include the nucleic acids described herein.

[0022] In certain non-limiting embodiments, the vector includes one or more AAV
inverted terminal repeat (ITR) sequences.

[0023] In certain non-limiting embodiments, the AAV ITR sequences include an ITR
sequence of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes, or a mixture of ITR
sequences therefrom.

[0024] In certain non-limiting embodiments, the AAV vector includes an AAV
capsid sequence.

[0025] In certain non-limiting embodiments, the AAV capsid sequence includes a VP1, VP2 and/or VP3 capsid sequence having at least 90% identity to the VP1, VP2 and/or VP3 sequences of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rhl 0, Rh74 or AAV-2i8.

[0026] Also disclosed are cells that produce AA V vectors of the presently disclosed inventive concepts.

[0027] Also disclosed are gene therapy methods of treating conditions related to retinal degeneration in mammalian subjects in need of such treatment, by administering to the subject a therapeutically-effective amount of a vector including a nucleic acid which encodes a mutant mammalian RPE65 protein or portion thereof, wherein the mutant mammalian RPE65 protein or portion thereof is expressed in vivo in retinal cells of the subject and has isomerohydrolase activity. The vector, nucleic acid and mutant mammalian RPE65 protein or portion thereof used in such gene therapy methods may include those described herein.

[0028] Also disclosed are methods of treating conditions related to retinal ,degeneration in subjects in need of such treatment, by administering to the subject a therapeutically-effective amount of at least one mutant mammalian RPE65 protein or a portion thereof described herein, thereby mitigating the condition related to retinal degeneration in the subject.

[0029] In certain non-limiting embodiments, the condition related to retinal degeneration is Leber's Congenital Amaurosis or Retinitis pigmentosa.

[0030] In certain non-limiting embodiments of the methods described herein, the subject treated is human.

[0031] In certain non-limiting embodiments of the methods described herein, the subject is less than 18 years old, less than 15 years old, less than 12 years old, less than 10 years old, between about 8-10 years old, between about 6-10 years old, between about 4-6 years old, between about 1-3 years old or is less than 1 year old or less.

[0032] In certain non-limiting embodiments of the methods described herein, the subject may have or be at risk of having retinal degeneration or dystrophy caused by mutation.

[0033] In certain non-limiting embodiments of the methods described herein, the vector employed may be administered to one or more eyes of the subject.

[0034] In certain non-limiting embodiments of the methods described herein, the vector employed may be administered to the subretinal space and/or suprachoroidal space of one or more eyes of the subject.

[0035] In certain non-limiting embodiments of the methods described herein, the vector employed is an AAV vector.

[0036] In certain non-limiting embodiments of the methods described herein, about 1x108 or more AAV vector genomes are administered to one or more eyes of the subject.

[0037] In certain non-limiting embodiments of the methods described herein, about 1 x108 to about 1 x1014 AAV vector genomes are administered to one or more eyes of the subject.

[0038] In certain non-limiting embodiments of the methods described herein, about 1 x109 to about 1 x1013AAV vector genomes are administered to one or more eyes of the subject.

[0039] In certain non-limiting embodiments of the methods described herein, the vector or mutant mammalian RPE65 protein or portion thereof is administered in a volume of about microliters (il) to about 1,000 1.

[0040] In certain non-limiting embodiments of the methods described herein, the vector or mutant mammalian RPE65 protein or portion thereof is administered in a volume of about 50 1A1 to about 800 IA.

[0041] In certain non-limiting embodiments of the methods described herein, the vector or mutant mammalian RPE65 protein or portion thereof is administered in a volume of about 100 ul to about 600

[0042] In certain non-limiting embodiments of the methods described herein, the vector or mutant mammalian RPE65 protein or portion thereof is administered in a volume of about 200 ul to about 500

[0043] In certain non-limiting embodiments of the methods described herein, a vector or mutant mammalian RPE65 protein or portion thereof described herein is delivered or introduce in cells of the retinal pigment epithelium of one or more eyes of the subject.

[0044] Certain embodiments of the presently disclosed inventive concepts include, but are not limited to, mutant mammalian RPE65 proteins or portions thereof that include an amino acid sequence having at least 90% identity with at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID
NO:15, and SEQ ID NO:17; and having at least one amino acid substitution that results in an about 2-fold increase to an about 6-fold increase in isomerohydrolase activity as compared to wild-type human RPE65.

[0045] In certain non-limiting embodiments, the about 2-fold to about 6-fold increase in isomerohydrolase activity is determined by in vitro isomerohydrolase activity assay.

[0046] In certain non-limiting embodiments, the amino acid substitution is Lys, Arg, or His at position 170; and/or Gly or Ala at position 297.

[0047] In certain non-limiting embodiments, the amino acid substitution(s) result in an about 3.2-fold increase in isomerohydrolase activity compared to wild-type human RPE65.

[0048] In certain non-limiting embodiments, the amino acid substitutions include a Lys, Arg, or His substitution at position 170; a Gly or Ala substitution at position 297; a Tyr, Phe, or His substitution at position 2; a Ser, Thr, or Cys substitution at position 3; and a Val, Ala, or Ile substitution at position 26.

[0049] In certain non-limiting embodiments, the amino acid substitutions result in an about 4.4 fold increase in isomerohydrolase activity compared to wild-type human RPE65.
BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Several embodiments of the presently disclosed inventive concepts are hereby illustrated in the appended drawings. It is to be noted however, that the appended drawings only illustrate several typical embodiments and are therefore not intended to be considered limiting of the scope of the presently disclosed inventive concepts. Further, in the appended drawings, like or identical reference numerals may be used to identify common or similar elements, and not all such elements may be so numbered. The figures are not necessarily to scale, and certain features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0051] FIG.1 shows an amino acid sequence alignment of wild type (wt) human (hRPE65) and chicken RPE65 (cRPE65). Amino acid residues of cRPE65 identical to those of hRPE65 are indicated as dot ".".

[0052] FIGS. 2A-2R show: (A) Amino acid sequence of wild type human RPE65 (SEQ

ID NO:1); (B) Nucleic acid sequence encoding human RPE65 (SEQ ID NO:2); (C) Amino acid sequence of chicken RPE65 (SEQ ID NO:3); (D). Nucleic acid sequence encoding chicken RPE65 (SEQ ID NO:4); (E) Amino acid sequence of crab-eating macaque (SEQ ID NO:5); (F) Nucleic acid sequence encoding crab-eating macaque RPE65 (SEQ ID
NO:6); (G) Amino acid sequence of green monkey RPE65 (SEQ ID NO:7); (H) Nucleic acid sequence encoding green monkey RPE65 (SEQ ID NO:8); (I) Amino acid sequence of bovine RPE65 (SEQ ID NO:9); (J) Nucleic acid sequence encoding bovine RPE65 (SEQ ID
NO:10); (K) Amino acid sequence of goat RPE65 (SEQ ID NO:11); (L) Nucleic acid sequence encoding goat RPE65 (SEQ ID NO:12); (M) Amino acid sequence of dog (SEQ ID NO:13); (N) Nucleic acid sequence encoding dog RPE65 (SEQ ID NO:14);
(0) Amino acid sequence of domestic cat RPE65 (SEQ ID NO:15); (P). Nucleic acid sequence encoding domestic cat RPE65 (SEQ ID NO:16); (Q) Amino acid sequence of rat (SEQ ID NO:17); and (R). Nucleic acid sequence encoding rat RPE65 (SEQ ID
NO:18).

[0053] FIGS.3A-3D show: Expression and enzymatic activities of RPE65 from humans and chicken. Plasmids expressing wt hRPE65, cRPE65 and red fluorescence protein (rfp, negative control) were separately transfected into 293A-LRAT cells. Expression levels and enzymatic activities of hRPE65 and cRPE65 were measured by Western blot analysis (A, Pc;
bovine RPE microsomal protein (2.5 tig), Nc; Rfp, Hum; hRPE65 and Chk; cRPE65, 20 u.g each) and in vitro isomerohydrolase activity assay, respectively. All-trans-CH] retinol (0.2 ;AM) was incubated with 125 ug of total cellular protein from the cells expressing rfp (B), hRPE65 (C) and cRPE65 (D) for 2 hr, and the generated retinoids were analyzed by HPLC. Peak 1, retinyl esters; 2, 11 cROL.

[0054] FIGS.4A-4G show: Impacts of site-directed mutations on isomerohydrolase activity and protein level of RPE65. Plasmids expressing wt hRPE65 and cRPE65, and the indicated hRPE65 mutants were separately transfected into 293A-LRAT cells and cultured for 48 hr. (A-D) The equal amount of total proteins from cell lysates (20 pg) was used for Western blotting using an antibody specific for RPE65, with an anti-f3-actin antibody as loading control (A; single, B; double, C; triple and quadruple mutants in the 1st experimental set, and D; single, E; double mutants in the 2nd experimental set. Pc;
positive control (bovine RPE microsomal protein, 2.5 [tg) Nc; negative control (red fluorescence protein), WT; wt hRPE65). RPE65 levels were semi-quantified by densitometry and normalized by (3-actin levels. (F and G) In vitro activity assays were performed with the same batch of samples of Western blot analyses. Isomerohydrolase activities were quantified by generated 11431-1]-cis retinol from 3 independent measurements and normalized by their relative RPE65 protein levels. Values were expressed as relative activity (% of wt hRPE65 activity, mean SEM, n=3). In the enzymatic activity tables (F and G), human (0) and chicken (0) residues were indicated as white and black circles, respectively. The vertical numbers indicate the positions of candidate residues. Letters above and below the panel in the tables (F and G) represent hRPE65 and cRPE65 amino acid residues at the indicated positions, respectively: ex. "= = o o"
in the table of 1st experimental set (F) indicates a triple mutant (T39R/N170K/Q497P) of hRPE65.

[0055] FIGS.5A-5C show: Schematic diagram of chimeric human RPE65 mutants and their enzymatic activities. (A) The positions of candidate sites in each fragment and specific restriction sites were represented in the diagram (white bar; hRPE65, gray bar; cRPE65, F1-F6; chimeric hRPE65 replaced with the corresponding cRPE65 fragment). (B) Expression levels of hRPE65, cRPE65 and chimeras were measured by Western blot analysis (Pc; 2.5 g of bovine RPE microsomal protein, 20 g of total cellular protein from Hum;
hRPE65, Chk;
cRPE65, and, F1-F6), and protein levels of RPE65 were semi-quantified by densitometry (C).
Enzymatic activities were measured by in vitro isomerohydrolase assay and quantified using generated 11431-1]-cis retinol and normalized by their relative RPE65 protein levels (C).

[0056] FIGS.6A-6B show: Impacts of the site-directed mutations and fragment replacement with those of cRPE65 on protein levels and enzymatic activities of hRPE65.
The identified site-directed mutants in this study (N170K and K297G) and the F1 chimera were combined to produce the mutant sIMH (F1/N170K/K297G). Th e identified point mutants and F1 chimera of RPE65 were expressed in 293A-LRAT cells. Cells were . harvested 48 hr after the transfection, and protein levels of RPE65 were confirmed by Western blot analyses (A). Enzymatic activities were measured by in vitro isomerohydrolase assay and quantified by generated 1143M-cis retinol (B). Isomerohydrolase activities were quantified by generated 1143M-cis retinol (picomole/hr) and presented as % of wt hRPE65 activity (mean SEM, n=3).

[0057] FIGS.7A-7C show: Analyses of catalytic efficiencies on wt hRPE65 and sIMH.
Adenoviruses expressing hRPE65 and sIMH were prepared. Wt hRPE65 and sIMH were separately expressed in 293A (without LRAT) cells for 24 hr at MOI 100 and its expressions were evaluated by Western blot analysis (A). Various concentrations of substrate in liposome with total cellular protein lysates (125 lig) were applied in in vitro enzyme assays and Km and Vmax of wt hRPE65 (B) and sIMH (C) were calculated through Michaelis-Menten plot of 11cROL generation, respectively.

[0058] FIGS.8A-8C show: Locations of Asn170 and Lys297, and the F1 fragment in an RPE65 3D structure model. The locations of two identified key residues and the Fl fragment are shown in the 3D model based on the crystal structure of bovine RPE65 (PDB
accession:
3FSN). The iron binding site, within the catalytic domain, is indicated by an orange dotted circle. The disordered segment (Phe109-Va1126), which contains a palmitylated Cys residue (Cys112), is shown by a pink dotted line. The entrance of the hydrophobic tunnel containing active site is indicated by a red dotted-circle. The location of the Fl fragment is indicated by the yellow dotted line. Both Asn170 and Lys297 residues are located on the surface of the protein (A and B) and more than 20A distant from iron-II in the potential catalytic domain (C).

[0059] FIG.9 shows non-limiting examples of alternate mutant substitutions in various positions of mammalian RPE65 sequences.
DETAILED DESCRIPTION

[0060] Before describing various embodiments of RPE65 mutants and methods of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description. The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning;
and the embodiments are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that the presently disclosed inventive concepts may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description.

[0061] Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed inventive concepts shall have the meanings that are commonly understood by those having ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0062] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the presently disclosed inventive concepts pertain. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0063] All of the compositions and methods of production and application thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the presently disclosed inventive concepts have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the inventive concepts. All such similar substitutes and modifications apparent to those having ordinary skill in the art are deemed to be within the spirit, scope, and concept of the inventive concepts as defined herein.

[0064] As utilized in accordance with the methods and compositions of the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0065] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."
The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or when the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or any integer inclusive therein. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y and Z" will be understood to include X alone, Y
alone, and Z
alone, as well as any combination of X, Y and Z.

[0066] As used in this specification and claim(s), the words "comprising"
(and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0067] The term "or combinations thereof' as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof' is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0068] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the composition, the method used to administer the composition, or the variation that exists among the study subjects.

[0069] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, the term "substantially" means that the subsequently described event or circumstance occurs at least 90% of the time, or at least 95% of the time, or at least 98% of the time.

[0070] The term "pharmaceutically acceptable" refers to compounds and compositions which are suitable for administration to humans and/or animals without undue adverse side effects such as toxicity, irritation and/or allergic response commensurate with a reasonable benefit/risk ratio.

[0071] By "biologically active" is meant the ability to modify the physiological system of an organism without reference to how the active agent has its physiological effects.

[0072] As used herein, "pure," or "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other object species in the composition thereof), and particularly a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80% of all macromolecular species present in the composition, more particularly more than about 85%, more than about 90%, more than about 95%, or more than about 99%. The term "pure" or "substantially pure" also refers to preparations where the object species (e.g., the peptide compound) is at least 60% (w/w) pure, or at least 70%
(w/w) pure, or at least 75% (w/w) pure, or at least 80% (w/w) pure, or at least 85% (w/w) pure, or at least 90% (w/w) pure, or at least 92% (w/w) pure, or at least 95%
(w/w) pure, or at least 96% (w/w) pure, or at least 97% (w/w) pure, or at least 98% (w/w) pure, or at least 99%
(w/w) pure, or 100% (w/w) pure.

[0073] The terms "subject" and "patient" are used interchangeably herein and will be understood to refer to a warm blooded animal, particularly a mammal. Non-limiting examples of animals within the scope and meaning of this term include dogs, cats, rats, mice, guinea pigs, horses, goats, cattle, sheep, zoo animals, Old and New World monkeys, non-human primates, and humans. In humans, both adults 18 years old and greater, and children less than 18 years old, are appropriate for treatment.

[0074] Treatment" refers to therapeutic treatments. The term "treating"
refers to administering the composition to a patient for therapeutic purposes.

[0075] "Prevention" refers to prophylactic or preventative treatment measures.

Administration to a subject can be performed prior to development of an adverse symptom, condition, complication, etc., caused by or associated with the disease. For example, a screen (e.g., genetic) can be used to identify candidate subjects for the compositions and methods of the presently disclosed inventive concepts. Such subjects therefore include those screened positive for an insufficient amount or a deficiency in a functional gene product (protein), or that produce an aberrant, partially functional or non-functional gene product (protein).

[0076] The terms "therapeutic composition" and "pharmaceutical composition"
refer to an active agent-containing composition that may be administered to a subject by any method known in the art or otherwise contemplated herein, wherein administration of the composition brings about a therapeutic effect as described elsewhere herein.
In addition, the compositions of the presently disclosed inventive concept may be designed to provide delayed, controlled, extended, and/or sustained release using formulation techniques which are well known in the art.

[0077] The term "effective amount" refers to an amount of an active agent which is sufficient to exhibit a detectable therapeutic effect, without excessive adverse side effects (such as substantial toxicity, irritation and allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of the inventive concepts. The therapeutic effect may include, for example but not by way of limitation, a partial or complete restoration of vision. For example, to assess a beneficial effect on retinal/visual function testing assays include, electroretinograms (ERGS), pupillometry, and behavioral testing (e.g., an obstacle course with variations in light), before and after treatment.

[0078] The effective amount for a patient will depend upon the type of patient, the patient's size and health, the nature and severity of the condition to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0079] The term "ameliorate" means a detectable or measurable improvement in a subject's disease or symptom thereof. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease.

[0080] A successful treatment outcome can lead to a "therapeutic effect,"
or "benefit" of decreasing, reducing, inhibiting, suppressing, limiting, controlling or preventing the occurrence, frequency, severity, progression, or duration of a disease, or consequences of the disease in a subject. Treatment methods and uses affecting one or more underlying causes of the disease or adverse symptoms are therefore considered to be beneficial. A
decrease or reduction in worsening, such as stabilizing the disease, is also a successful treatment outcome.

[0081] A therapeutic benefit therefore need not be complete ablation or reversal of the disease, or any one, most or all adverse symptoms, complications, consequences or underlying causes associated with the disease. Thus, a satisfactory endpoint is achieved when there is an incremental improvement such as a partial decrease, reduction, inhibition, suppression, limit, control or prevention in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal of the disease (e.g., stabilizing), over a short or long duration of time (hours, days, weeks, months, etc.). Effectiveness of a method or use, such as a treatment that provides a potential therapeutic benefit or improvement of a disease, can be ascertained by various methods, such as retinal/visual function testing assays including electroretinograms (ERGS), pupillometry, and behavioral testing after treatment, etc.

[0082] The term "homologous" or "% identity" as used herein means a nucleic acid (or fragment thereof) or a protein (or a fragment thereof) having a degree of homology to the corresponding natural reference nucleic acid or protein that may be in excess of 70%, or in excess of 80%, or in excess of 85%, or in excess of 90%, or in excess of 91%, or in excess of 92%, or in excess of 93%, or in excess of 94%, or in excess of 95%, or in excess of 96%, or in excess of 97%, or in excess of 98%, or in excess of 99%. For example, in regard to peptides or polypeptides, the percentage of homology or identity as described herein is typically calculated as the percentage of amino acid residues found in the smaller of the two sequences which align with identical amino acid residues in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to assist in that alignment (as set forth by Dayhoff in Atlas of Protein Sequence and Structure (1972) Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C.). In one embodiment, the percentage homology as described above is calculated as the percentage of the components found in the smaller of the two sequences that may also be found in the larger of the two sequences (with the introduction of gaps), with a component being defined as a sequence of four, contiguous amino acids. Also included as substantially homologous is any protein product which may be isolated by virtue of cross-reactivity with antibodies to the native protein product. Sequence identity or homology can be determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms. A non-limiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul (1990) Proc.
Natl. Acad.
Sci. USA, 87, 2264-2268, modified as in Karlin & Altschul (1993) Proc. Natl.
Acad. Sci.
USA, 90, 5873-5877.

[0083] In one embodiment "% identity" represents the number of amino acids or nucleotides which are identical at corresponding positions in two sequences of a protein having the same activity or encoding similar proteins. For example, two amino acid sequences each having 100 residues will have 95% identity when 95 of the amino acids at corresponding positions are the same.

[0084] Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller (1988) CABIOS, 4, 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman (1988) Proc. Natl.
Acad. Sci. USA, 85, 2444-2448.

[0085] Another algorithm is the WU-BLAST (Washington University BLAST) version 2.0 software (WU-BLAST version 2.0 executable programs for several UNIX
platforms).
This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish (1996) Local alignment statistics. In Methods in Enzymology (R. Doolittle, ed.) 266, 460-480; Altschul et al. (1990) Journal of Molecular Biology, 215, 403-410; Gish & States (1993) Nature Genetics, 3: 266-272; Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA, 90, 5873-5877; all of which are incorporated by reference herein).

[0086] In addition to those otherwise mentioned herein, mention is made also of the programs BLAST, gapped BLAST, BLASTN, BLASTP, and PSI-BLAST, provided by the National Center for Biotechnology Information. These programs are widely used in the art for this purpose and can align homologous regions of two amino acid sequences.
In all search programs in the suite, the gapped alignment routines are integral to the database search itself. Gapping can be turned off if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer.
The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer. Any combination of values for Q
and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.

[0087] The terms "polynucleotide sequence" or "nucleic acid," as used herein, include any polynucleotide sequence which encodes a mutant RPE65 protein product including polynucleotides in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The DNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
The polynucleotide sequence encoding a mutant protein, or encoding a fragment of a mutant protein can be substantially the same as the coding sequence of the endogenous coding sequence as long as it encodes a biologically active mutant protein product. Further, the mutant protein, or fragment of a mutant protein may be expressed using polynucleotide sequence(s) which differ in codon usage due to the degeneracies of the genetic code or allelic variations.

[0088] The terms "infection," "transduction," and "transfection" are used interchangeably herein and mean introduction of a gene, nucleic acid, or polynucleotide sequence into cells such that the encoded protein product is expressed. The polynucleotides of the presently disclosed inventive concepts may comprise additional sequences, such as additional coding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, transcription terminators, polyadenylation sites, additional transcription units under control of the same or different promoters, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of the presently disclosed inventive concepts.

[0089] The term "gene therapy" as used herein means genetic modification of cells by the introduction of exogenous DNA or RNA into these cells for the purpose of expressing or replicating one or more peptides, polypeptides, proteins, oligonucleotides, or polynucleotides in vivo for the treatment or prevention of disease or deficiencies in humans or animals. Gene therapy is generally disclosed in U.S. Pat. No. 5,399,346. Any suitable route of administration of the nucleic acid or protein may be employed for providing a subject with pharmaceutical compositions of the presently disclosed inventive concepts. For example, parenteral (subcutaneous, subretinal, suprachoroidal, intramuscular, intravenous, transdermal) and like forms of administration may be employed. Dosage formulations include injections, implants, or other known and effective gene therapy delivery methods.

[0090]
Genetic delivery vehicles (vectors) for the nucleic acids encoding a protein product of the presently disclosed inventive concepts include any vector suitable for promoting expression of the mutant RPE65 protein product and may optionally comprise an operatively attached promoter sequence which is specific for retinal pigment epithelial cells (e.g., see U.S. Patent No. 8,785,413). Such gene delivery vehicles are well known in the art to persons having ordinary skill in the art; thus, their detailed description is not deemed necessary herein. For example, a nucleic acid encoding a protein product of the presently disclosed inventive concepts may be contained in adeno-associated virus vectors (e.g., as disclosed in U.S. Pat. Nos. 5,139,941, 5,436,146, and 5,622,856), an attenuated or gutless adenoviral vectors, (e.g., as disclosed in Morsy, M. A. and Caskey, C. T. (1999) Mol.
Med. Today 5:18-24; and U.S. Pat. No. 5,935,935), lentiviral vectors (such as are disclosed in U.S. Pat.

Nos. 5,665,577; 5,994,136; and 6,013,516), plasmids or synthetic (non-viral) vectors (such as disclosed in U.S. Pat. Nos. 4,394,448 and 5,676,954), and/or nanoparticles (such as disclosed, for example, in U.S. Patents 6,217,912; 7514098; and 8,323,618). Alternative viral vectors include, but are not limited to, retroviral vectors (such as are disclosed in U.S. Pat. Nos.
5,672,510; 5,707,865; and 5,817,491), herpes virus vectors (such as are disclosed in U.S. Pat.
No. 5,288,641), and sindbis virus vectors and papilloma virus vectors (such as are disclosed in EP 820 773). The vectors may be either monocistronic, bicistronic, or multicistronic.

[0091] An adenoviral vector may include essentially the complete adenoviral genome (Shenk, et al. (1984) Curr. Top. Microbiol. Immunol., 111:1-39).
Alternatively, the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted. Adenoviral vectors may be produced according to He, et al. (1998) PNAS, 95:2590-2514; Chartier, et al. (1996) J. Virol., 70:4805-4810; and Hitt, et al. (1995) Methods in Molecular Genetics, 7:13-30. Methods of transferring genes into cells using adenoviral vectors have been described in PCT/US95/15947. A
number of adenoviral vectors have been developed for the transduction of genes into cells (Berkner, et al. (1988) BioTechniques 6:616-629). Constitutive high level expression of the transduced gene products has been achieved. Th ese vectors have the inherent advantage over the retroviral vectors in not requiring replicating cells for infection, making them suitable vectors for somatic gene therapy (Mulligan, R. C. (1993) Science 260:926-932). The feasibility for transducing genes associated with glucose metabolism, using adenovirus-mediated transfer in primary rat hepatocytes and myoblast in culture, has been described (Baque, et al. (1994) Biochem. J. 304 (Pt 3):1009-1014; Gomez-Foix, et al. (1992) J. Biol. Chem.
267:25129-25134).

[0092] Vectors which may be used in the methods of the presently disclosed inventive concepts are also described in Narfstrom, et al. (2003) "Functional and structural recovery of the retina after gene therapy in the RPE65 null mutation dog." Invest.
Ophthalmol. Vis. Sci.
44, 1663-1672; Bennett, et al. (1994) "Adenovirus vector-mediated in vivo gene transfer into adult murine retina." Invest. Ophthalmol. Vis. Sci., 35, 2535-2542; Zhang, et al. (2008) "Distinctive gene transduction efficiencies of commonly used viral vectors in the retina."
Curr. Eye Res. 33, 81-90; Weber, et al. (2003) "Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery." Mol. Ther., 7, 774-781;
Yokoi, et al. (2007) "Ocular gene transfer with self-complementary AAV
vectors." Invest.
Ophthalmol. Vis. Sci., 48, 3324-3328; Auricchio, et al. (2001) "Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics:
the retina as a model." Hum. Mol. Genet., 10, 3075-3081; Pang, et al. (2008) "Comparative analysis of in vivo and in vitro AAV vector transduction in the neonatal mouse retina:
effects of serotype and site of administration." Vision Res., 48, 377-385; Yanez-Munoz, et al., (2006) "Effective gene therapy with nonintegrating lentiviral vectors." Nat. Med., 12, 348-353;
Bemelmans, et al. (2006) "Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis." PLoS Med., 3, 1892-1903;
Miyoshi, et al. (1997) "Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector." Proc. Natl. Acad. Sci. USA, 94, 10319-10323; Farjo, et al.
(2006) Efficient non-viral ocular gene transfer with compacted DNA nanopatlicles. PLoS One, 1, e38;
Kachi, et al. (2005) "Nonviral ocular gene transfer." Gene Ther., 12, 843-851;
and Johnson, et al. (2008) "Technical brief: subretinal injection and electroporation into adult mouse eyes."
Mol. Vis., 14, 2211-2226.

[0093] A recombinant vector (e.g., lenti-, parvo-, AAV) sequence can be packaged-referred to herein as a "particle" for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant vector sequence is encapsidated or packaged into an AAV particle, the particle can also be referred to as a "rAAV." Such particles include proteins that encapsidate or package the vector genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins.

[0094] A vector "genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., AAV) particle.
In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non vector genome portion of the recombinant plasmid is referred to as the "plasmid backbone," which is important for cloning and amplification of the plasmid, a process that is needed for recombinant virus production, but is not itself packaged or encapsidated into virus (e.g., AAV) particles.

[0095] Thus, a vector "genome" refers to the portion of the vector plasmid that is packaged or encapsidated (e.g., AAV), and which contains a sequence that encodes a mutant RPE65 protein or fragment thereof The non-vector genome portion of the recombinant plasmid is the "plasmid backbone" that is important for cloning and amplification of the plasmid, e.g., has a selectable marker, such as Kanamycin, but is not itself packaged or encapsidated by virus (e.g., AAV).

[0096] AAV vectors can be any serotype. Representative serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rhl 0, Rh74 or AAV-2i8.

[0097] Vectors can include additional nucleic acid or protein elements. For example, an AAV vector can include one or two inverted terminal repeat (ITR) sequences of AAV
genome retained in the AAV vector. ITR sequences can comprise or be based upon ITRs from any AAV serotype, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rhl 0, Rh74 or AAV-2i8. Typically, in an AAV vector the nucleic acid encoding mutant RPE65 protein or fragment (portion) thereof is flanked by 5 and/or 3' AAV ITR sequences.

[0098] Additional non-limiting examples of nucleic acid sequences include expression control elements (e.g., a promoter, enhancer), introns, poly-Adenine sequence, stop codon, etc. Such sequences including expression control elements can be located at the 5' (i.e., "upstream"), 3' end (i.e., "downstream") of the transcribed sequence or within the sequence (e.g., in an intron). Such sequences including expression control elements can be located adjacent to or at a distance away from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100 to 500, or more nucleotides from the polynucleotide), even at considerable distances.

[0099] A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence (a nucleotide sequence that encodes a mutant RPE65 protein or fragment thereof). Another example of an expression control element is an enhancer, which can be located 5' and/or 3' of the transcribed sequence, or within the transcribed sequence.

[0100] A "promoter" can refer to a nucleic acid (e.g., DNA) sequence that is located adjacent to a sequence, such as a nucleotide sequence that encodes a mutant RPE65 protein or fragment thereof. A promoter typically increases the amount of nucleic acid expressed to which it is operatively linked as compared to an amount expressed when no promoter exists.
An "enhancer" can refer to a sequence that is located adjacent to the sequence, such as a nucleotide sequence that encodes a mutant RPE65 protein or fragment thereof.
Enhancer elements are typically located upstream of a promoter element but also function and can be located downstream of a promoter, or be within a DNA sequence. Hence, an enhancer element can be located 10-25, 25-50, 50-100 100-200, 200-300 or more base pairs upstream or downstream of a sequence that encodes a mutant RPE65 protein or fragment thereof Enhancer elements typically also increase expression of an operatively linked sequence.

[0101] As used herein, the term "operable linkage" or "operably linked"
refers to a physical or functional juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. More specifically, for example, two DNA
sequences operably linked means that the two DNAs are arranged (cis or trans) in such a relationship that at least one of the DNA sequences is able to exert a physiological effect upon the other sequence.

[0102] Any suitable retinal pigment epithelial-specific promoter sequence may be used with the mutant RPE65 protein-encoding or fragment-encoding nucleic acids.
Selection of the promoter to be employed may be made from among a wide number of native, constitutive or inducible promoters that can express the RPE65 protein-encoding or fragment-encoding nucleic acid, for example, in an ocular context.

[0103] In one embodiment, a promoter is cell-specific. The term "cell-specific" means that the particular promoter selected for the recombinant vector can direct expression of the selected transgene is a particular ocular cell type. As one example, the promoter is specific for expression in RPE cells. As another example, a promoter is specific for expression in photoreceptor cells.

[0104] Representative non-limiting examples of RPE-specific promoters include the RPE-65 promoter, the tissue inhibitor of metalloproteinase 3 (Timp3) promoter, and the tyrosinase promoter. Still other RPE-specific promoters are known in the art (See, e.g., promoters described in International Patent Publication No. WO 00/15822).

[0105] Representative non-limiting examples of photoreceptor promoters include the rod opsin promoter, the red-green opsin promoter, the blue opsin promoter, the inter photoreceptor binding protein (IRBP) promoter and the cGMP-.beta.-phosphodiesterase promoter (See, e.g., the promoters described in International Patent Publication No. WO
98/48097.

[0106] Expression control elements include ubiquitous or promiscuous promoters/enhancers which are capable of driving expression in many different cell types. In a particular embodiment, a promoter is constitutive. Representative non-limiting examples of constitutive promoters include cytomegalovirus (CMV) immediate early enhancer/chicken 13-actin (CPA) promoter-exon 1-intron 1 element, Rous sarcoma virus (RSV) promoter/enhancer sequences, LTR promoter/enhancer, SV40 promoter, CMV
promoter, dihydrofolate reductase promoter, and phosphoglycerol kinase (PGK) promoter.

[0107] AAV vectors can include capsids of any serotype. Representative AAV
capsid serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8. AAV vectors can include capsid chimers or variants of any serotype. Particular AAV capsid variants include capsid variants of such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rhl 0, Rh74 or AAV-2i8, such as a capsid sequence with an amino acid substitution, deletion or insertion/addition.

[0108] In certain embodiments of the presently disclosed inventive concepts, compositions comprising a mutant RPE65 protein (and/or a therapeutically effective fragment thereof) are provided in a therapeutic treatment. Alternatively, compositions comprising a nucleotide sequence encoding the mutant RPE65 protein or fragment may be provided in a gene therapy treatment. Also provided are methods of producing these compositions, along with methods of use thereof. In one embodiment, the compositions are utilized for treatment of retinal degeneration-related diseases (dystrophies) or conditions in a subject, including but not limited to Leber's Congenital Amaurosis and Retinitis Pigmentosa (e.g., autosomal recessive retinitis pigmentosa). As noted, a particular treatment method is by gene therapy, for example via adeno-associated virus, lentivirus, plasmid, or nanoparticle.
In certain embodiments, the treatment results in a reduction in the occurrence and/or severity of vision loss in a mammalian subject such as a human.

[0109] In certain embodiments, the treatments of the presently disclosed inventive concepts use a pharmaceutical composition comprising a vector that expresses the desired mutant RPE65 protein or fragment in vivo under appropriate or suitable conditions or in a suitable host cell. The pharmaceutical compositions can comprise, consist essentially of, or consist of one or more vectors, e.g., expression vectors (viral such as AAV, lenti, etc.), such as in vivo expression vectors, comprising, consisting essentially of, or consisting of and expressing a nucleic acid encoding a mutant RPE65 protein or fragment in combination with a pharmaceutically acceptable carrier, excipient, and/or vehicle. In certain embodiments, the vector comprises, consists essentially of, or consists of and expresses at least one nucleic acid encoding a mutant RPE65 protein or fragment, in a pharmaceutically acceptable carrier, excipient, and/or vehicle. Thus, according to an embodiment of the presently disclosed inventive concepts, the other vector(s) in the composition comprises a nucleic acid encoding a mutant RPE65 protein or fragment.

[0110] The pharmaceutically acceptable carrier, vehicle, and/or excipient facilitates transfection and/or improves preservation of the vector. Any pharmaceutically acceptable carriers, vehicles, and excipients known in the art or otherwise contemplated herein may be utilized in accordance with the presently disclosed inventive concepts. For example but not by way of limitation, a pharmaceutically acceptable carrier, vehicle, or excipient can be water, a 0.9% NaC1 (e.g., saline) solution, or a phosphate buffer. Other pharmaceutically acceptable carriers, vehicles, and excipients that can be used in the methods of the presently disclosed inventive concepts include, but are not limited to, poly(L-glutamate) or polyvinylpyrrolidone. The pharmaceutically acceptable carrier, vehicle, or excipient may be any compound or combination of compounds facilitating the administration of the vector, increasing the level of expression, and/or increasing the duration of expression.

[0111] Doses and dose volumes are discussed herein in the general description and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue experimentation. For example, the dose volumes can be between about 0.01 and about 2 ml, such as between about 0.02 and about 1 ml. More particular dose volumes can be between about 0.05 and about 0.80 ml, between about 0.10 and about 0.60 ml, or between about 0.20 and about 0.50 ml.

[0112] The therapeutic and/or pharmaceutical compositions, in non-limiting embodiments, contain viral particles per dose in a range of, for example, from about 104 to about 1011 particles, from about 105 to about 101 particles, or from about 106 to about 109 particles. In the context of AAV vectors, vector genomes are provided in in a range of, for example, from about 104 to about 1014 vector genomes, from about 105 to about 1013 vector genomes, from about 106 to about 1013 vector genomes, from about 107 to about 1013 vector genomes, from about 108 to about 1013 vector genomes, or from about 109 to about 1013 vector genomes. Such doses/quantities of AAV vector are useful in the methods set forth herein.

[0113] The presently disclosed inventive concepts contemplate at least one administration to a subject of an efficient amount of the therapeutic composition made according to the presently disclosed inventive concepts. This administration may be via various routes including, but not limited to, intramuscular, subcutaneous, intraocular, subconjunctival, subretinal, suprachoroidal, or intravascular.

[0114] By way of illustration, mutant RPE65 protein or fragments that are encompassed by the proteins/nucleic acids of the presently disclosed inventive concepts include, but are not limited to, mutant RPE65 protein or fragments that are encoded by nucleotide sequences that are not exactly the same as the nucleotide sequences disclosed herein, but wherein the changes in the nucleotide sequences do not change the encoded amino acid sequence, or merely result in conservative substitutions of amino acid residues, deletion and/or addition of one or a few amino acids, substitution of amino acid residues by amino acid analogs that do not significantly affect the properties of the encoded polypeptides, and the like. Examples of conservative amino acid substitutions include, but are not limited to, glycine/alanine substitutions; valine/isoleucine/leucine substitutions; asparagine/glutamine substitutions;
aspartic acid/glutamic acid substitutions; serine/threonine/methionine substitutions;
lysine/arginine/histidine substitutions; and phenylalanine/tyrosine/tryptophan substitutions.

[0115] Accordingly, also included in the presently disclosed inventive concepts are mutant RPE65 proteins and fragments which at least one amino acid residue been removed and a different residue inserted in its place. For a detailed description of protein chemistry and structure, see Schulz, G. E. et al. (1979) Principles of Protein Structure, Springer-Verlag, New York, and Creighton, T. E. (1984) Proteins: Structure and Molecular Principles, W. H.
Freeman & Co., San Francisco. Types of substitutions which may be made are conservative substitutions and are defined herein as exchanges within one of the following groups:
(1) Small aliphatic, nonpolar or slightly polar residues: e.g., Ala, Ser, Thr, Gly; (2) Polar, negatively charged residues and their amides: e.g., Asp, Asn, Glu, Gin; and (3) Polar, positively charged residues: e.g., His, Arg, Lys. Pro, because of its unusual geometry, tightly constrains the chain. Substantial changes in functional properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above groups (or two other amino acid groups not shown above), which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Most substitutions according to the presently disclosed inventive concepts are those which do not produce radical changes in the characteristics of the protein. Even when it is difficult to predict the exact effect of a substitution in advance of doing so, one skilled in the art will appreciate that the effect can be evaluated by routine screening assays, such as an activity assay set forth herein (e.g., the isomerohydrolase activity assay).
Modifications of peptide properties including redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods known to the skilled artisan.

[0116] Other types of substitutions, variations, additions, deletions and derivatives that result in functional mutant RPE65 protein or fragments and homologs, as described above, are also encompassed by the presently disclosed inventive concepts, and one of skill in the art would readily know how to make, identify, or select such variants or derivatives, and how to test for RPE65 activity of those variants or derivatives. One of ordinary skill in the art may optimize the expression of the mutant RPE65 protein or fragments polypeptides of the presently disclosed inventive concepts to improve expression by any methods known in the art, including but not limited to, by removing cryptic splice sites, by adapting the codon usage by introducing a Kozak consensus sequence before the start codon, by changing the codon usage, or any combination thereof

[0117] In certain embodiments, the presently disclosed inventive concepts comprise a nucleic acid variant having identity or homology of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to at least one or more of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18, and which encodes a mutant RPE65 protein having identity or homology of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a polypeptide encoded by SEQ ID
NO:1, SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17, and which includes at least one or more amino acid substitutions described herein, such as one or more of those described in FIG.9.

[0118] In some embodiments, the DNA encoding the mutant RPE65 protein or fragment is a DNA which hybridizes with a wild type DNA described herein (e.g., SEQ ID
NO:2, SEQ
ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18) under stringent conditions. By "DNA which hybridizes under stringent conditions" is meant DNA obtained by colony hybridization, plaque hybridization, or Southern blot hybridization using DNA encoding RPE65 protein, specifically including DNA identified after hybridization, using a filter on which colony- or plaque-derived DNA has been immobilized in the presence of 0.7 to 1.0 M NaC1 at 65 C and washing the resulting filter using 0.1x to 2x SSC solutions (the composition of lx SSC
solution comprises 150 mM sodium chloride and 15 mM sodium citrate) at 65 C.
Hybridization can be carried out according to a method described, for example, in Molecular Cloning, A Laboratory Manual, the 2nd edition (Sambrook, Fritsch, & Maniatis eds., Cold Spring Harbor Laboratory Press, 1989).

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently disclosed inventive concepts belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed inventive concepts, suitable methods and materials are described herein.

[0120] Abbreviations used herein include: 11cROL, 11-cis retinol; AD, adenovirus; BTP, 1,3-bis[tris(hydroxymethyl)-methylamino] propane; cRPE65, chicken RPE65; HPLC, High-performance liquid chromatography; hRPE65, human RPE65; LCA, Leber's Congenital Amarousis; PVDF, Polyvinylidene fluoride; rAAV, recombinant adeno-associated virus;
RFP, red-fluorescent protein; RPE, retinal pigment epithelium; RPE65, RPE
specific 65-kDa protein; RP, Retinitis Pigmentosa; TBST, Tris-buffered saline with 0.1% Tween-20; wt, wild-type.

[0121] All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety.
In case of conflict, the specification, including definitions, will control.

[0122] All of the features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving a same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, disclosed features (e.g., nucleic acid, vector, plasmid, a recombinant vector (e.g., rAAV), vector genome, or virus particle) are an example of a genus of equivalent or similar features.

[0123] As noted above, as used herein, the singular forms "a", "and," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of such nucleic acids, reference to "a vector" includes a plurality of such vectors, and reference to "a virus" or "particle" includes a plurality of such virions/particles.

[0124] As used herein, all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to 80% or more identity, includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so forth.

[0125] Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10 includes 9, 8, 7, etc. all the way down to the number one (1).

[0126] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth.

Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth.

[0127] Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000, includes ranges of 1-20, 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000, etc.

[0128] The presently disclosed inventive concepts will be more readily understood by reference to the following examples and embodiments, which are included merely for purposes of illustration of certain aspects and embodiments of the presently disclosed inventive concepts, and are not intended to be limiting. The following detailed examples and methods describe how to make and use the various mutant proteins of the presently disclosed inventive concepts and are to be construed, as noted above, only as illustrative, and not limitations of the disclosure in any way whatsoever. Those skilled in the art will promptly recognize appropriate variations from the materials and procedures described herein.

[0129] Cone-dominant chicken RPE65 (cRPE65 - SEQ ID NO:3) is known to possess substantially greater isomerohydrolase activity than that of human RPE65 (hRPE65-SEQ ID
NO:1), bovine RPE65 (bRPE65 - SEQ ID NO:9) or other mammalian RPE65 protein.
As explained in detail below, the enzymatic activities of cRPE65, hRPE65 and mutants of hRPE65 were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants of the presently disclosed inventive concepts analyzed herein, two single point mutants, N170K and K297G, and a double mutant N170K/K297G of hRPE65 exhibited significantly higher catalytic activity than that of wt hRPE65. Further, when an amino terminal fragment (amino acids 1 met-33 Arg, ) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding n-terminal cRPE65 33-residue fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This highly efficient isomerohydrolase mutant combination (S2Y/I3S/L26V/N170K/K297G-further described below) can be used, in certain RPE65 gene therapy embodiments of the present disclosure to improve vision in mammalian subjects having or at risk of having retinal degeneration caused by RPE65 mutations, including but not limited to, humans.

[0130] In the work presently disclosed, using site-directed mutagenesis, a number of residues of hRPE65 were substituted with their positional counterparts of cRPE65 (alignment shown in FIG.1) and evaluated the isomerohydrolase activities of the generated hRPE65 mutants. The results are shown and discussed below.

[0131] EXPERIMENTAL PROCEDURES

[0132] Site-directed mutagenesis.

[0133] cDNAs encoding Wild-type (wt) hRPE65 protein (SEQ ID NO:1) and cRPE65 protein (SEQ ID NO:3) were subcloned into cloning vectors as described previously (Moiseyev, et al. (2005) "RPE65 Is the Isomerohydrolase in the Retinoid Visual Cycle."
Proc. Natl. Acad. Sci. USA, 102, 12413-12418; and Moiseyev, et al. (2008) "RPE65 from Cone-dominant Chicken Is a More Efficient Isomerohydrolase Compared with That from Rod-dominant Species." J. Biol. Chem., 283, 8110-8117). Selected residues in hRPE65 were replaced by their counterparts in cRPE65 using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's protocol. The introduced mutations were confirmed by sequencing from both strands using ABI-3730 DNA
sequencer (Applied Biosystems, Foster City, CA) and subcloned into an expression vector, pcDNA3.1 (-) (Invitrogen, Carlsbad, CA). Following the sequence confirmation, the expression constructs were purified by QIAfilter Maxi Prep kit (Qiagen, Valencia, CA).
Further, the hRPE65 cDNA (SEQ ID NO:2) and cRPE65 cDNA (SEQ ID NO:4) were individually subcloned into pUC18. To generate 6 restriction fragments of hRPE65 and cRPE65, unique restriction enzyme sites were introduced without changing amino acid sequence (see FIG.5A). Each fragment of hRPE65 was replaced by its counterpart of cRPE65 using the introduced restriction enzyme sites. All primer sets (SEQ ID NOS:19-42) used in this study are shown in Table 1.

[0134] Table 1: Primer sets.
Name Sequence SEQ M NO.
Hum T3 9R-Fwd 5'-CCCCCTCTGGCTCCGCGGCAGTCTCCTTC-3' SEQ ID NO:19 Hum T39R-Rev 51-GAAGGAGACTGCCGCGGAGCCAGAGGGGG-3 SEQ ID NO:20 Hum N170K-Fwd 5'-GGTTGATCTTTGCAAGTATGTCTCTGTC-3' SEQ ID NO:21 Hum N170K-Rev 5'-GACAGAGACATACTTGCAAAGATCAACC-3' SEQ ID NO:22 Hum C33 OT-Fwd 5 '-GATTGTGGATCTCTGCACCTGGAAAGGATTTG-3' SEQ ID NO:23 Hum C330T-Rev 5'-CAAATCCTTTCCAGGTGCAGAGATCCACAATC-3' SEQ ID NO:24 Hum Q497P-Fwd 5'-GCCCAGGAGCAGGACCAAAGCCTGCTTATC-3' SEQ ID NO:25 Hum Q497P-Rev 5'-GATAAGCAGGCTTTGGTCCTGCTCCTGGGC-3' SEQ ID NO:26 Hum C106Y-Fwd 5'-CAGAATTTGGCACCTATGCTTTCCCAGATCCC-3' SEQ ID NO:27 Hum C106Y-Rev 5'-GGGATCTGGGAAAGCATAGGTGCCAAATTCTG-3' SEQ ID NO:28 5'-GCTGACAAAAAAAGGGGAAAGTACCTCAATAATAAATACAG- SEQ ID NO:29 Hum K297G-Fwd 3' Hum K297G-Rev 5'-CTGTATTTATTATTGAGGTACTTTCCCCTTTTTTTGTCAGC-3' SEQ ID NO:30 Hum L510M-Fwd 5'-CTGAATGCCAAGGACATGAGTGAAGTTGCCCGG-3' SEQ ID NO:31 Hum L510M-Rev 5'-CCGGGCAACTTCACTCATGTCCTTGGCATTCAG-3' SEQ ID NO:32 Hum S53 3A-Fwd 5'-GGACTGTTCAAAAAAGCTTGAGCATACTCCAGCAAGC-3 SEQ ID NO: 33 Hum S533A-Rev 5'-GCTTGCTGGAGTATGCTCAAGCTTTTTTGAACAGTCC-3' SEQ ID NO: 34 Hum KpnI-Fwd 5'-CAGAATTTGGTACCTGTGCTTTCCCAG-3' SEQ ID NO:35 Hum KpnI-Rev 5'-CTGGGAAAGCACAGGTACCAAATTCTG-3' SEQ ID NO:36 Hum SaII-Fwd 5'-GGGTTTCTGATTGTCGACCTCTGCTGCTGG -3' SEQ ID NO:37 Hum SalI-Rev 5'-CCAGCAGCAGAGGTCGACAATCAGAAACCC-3' SEQ ID NO:38 ChkPstI-Fwd 5'-GCAGTTCCCCTGCAGTGACAGATTTAAG-3' SEQ ID NO:39 ChkPstI-Rev 5'-CTTAAATCTGTCACTGCAGGGGAACTGC-3' SEQ ID NO:40 Chk SmaI-Fwd 5'-GTGAAGTGGCCCGGGCAGAAGTGGAGG-3' SEQ ID NO:41 Chk SmaI-Rev 5'-CCTCCACTTCTGCCCGGGCCACTTCAC-3' SEQ ID NO :42

[0135] Plasmid transfection.

[0136] Constructed plasmids expressing wt hRPE65, cRPE65, and the hRPE65 mutants were purified using a QIAfilter Maxi Prep kit (Qiagen, Valencia, CA) and transfected into 293A-LRAT cells, a cell line stably expressing human LRAT (45), using Fugene 6 transfection reagent (Roche, Indianapolis, IN) or polyethylenimine (PEI, Polysciences, Inc., Warrington, PA; 1 mg/ml, pH 7.4, 2:1 of PEI:DNA ratio), and the culture media were replaced at 6 hr following the transfection. At 48 hr post transfection, cells were harvested by cell scraper and rinsed twice with ice-cold PBS. Protein levels and enzymatic activities of wt hRPE65 and its mutants were confirmed by Western blot analyses and in vitro isomerohydrolase activity assay.

[0137] Western blot analysis.

[0138] Total cellular protein concentrations were measured using a Bradford assay. Equal amounts of total cellular proteins (20 p.g) of 293A-LRAT cells expressing wt hRPE65 and cRPE65, and hRPE65 mutants and bovine RPE microsomal proteins (2.5 jig) as a positive control were resolved by electrophoresis through 8% Tris-glycine SDS
polyacrylamide gel and electrotransferred onto an immobilon PVDF membrane (Millipore, Billerica, MA) unless specified. The membrane was blocked with 5% (wt/vol) non-fat dry milk in TBST
(Tris-buffered saline with 0.1% Tween-20) for 30 min and subsequently incubated overnight at 4 C
with a 1:1,000 dilution of an anti-RPE65 polyclonal antibody (e.g., see Ma, et al. (2001) "Expression, purification, and MALDI analysis of RPE65." Invest. Ophthalmol.
Vis. Sci., 42, 1429-1435) to identify the key residues and 1:50,000 dilution of an anti-f3-actin monoclonal antibody (Sigma-Aldrich, St. Louis, MO). We used another RPE65 antibody (Moiseyev, et al. (2008) "RPE65 from Cone-dominant Chicken Is a More Efficient Isomerohydrolase Compared with That from Rod-dominant Species." J. Biol.
Chem., 283, 8110-8117) to compare the expression levels of cRPE65 and chimeric mutants of RPE65 to avoid the immunoreactivity difference on fragment 3. After four washes with TBST, the membrane was incubated in a light shielding container for 1.5 hr with 1:25,000 dilution of goat anti-mouse IgG conjugated with DyLight-549 and goat anti-rabbit IgG
conjugated with DyLight-649 (Pierce, Rockford, IL), and the bands were detected using FluorChem Q
imaging system (AlphaInnotech, San Leandro, CA). The signal intensities were semi-quantified by densitometry using AlphaView Q software (AlphaInnotech, San Leandro, CA), and averaged from at least 3 independent experiments.

[0139] Isomerohydrolase activity assay.

[0140] 293A-LRAT cells were separately transfected with plasmids expressing wt human and chicken RPE65 and hRPE65 mutants. 293A-LRAT cells expressing red fluorescence protein (rfp) were used as a negative control. Cells were lysed by sonication in a reaction buffer (10 mM 1,3-bis[tris(hydroxymethyp-methylamino] propane (BTP), pH 8.0, 100 mM
NaC1). All-trans [11,12-31-1]-retinol (1 mCi/ml, 45.5 Ci/mmol, American Radiolabeled Chemical, Inc., St. Louis, MO) in N,N-dimethyl formamide was used as the substrate for the isomerohydrolase assay. For each reaction, total cellular proteins (125 lig) were added into 200 1 of reaction buffer (10 mM BTP, pH 8.0, 100 mM NaC1) containing 0.2 1.11\4 of all-trans retinol, 1% BSA and 25 i_iM of cellular retinal aldehyde-binding protein. The reaction was stopped, and retinoids were extracted with 300 [d. of cold methanol and 300 j1.1 of hexane.
The generated retinoids were analyzed by normal phase HPLC. The peak of each retinoid isomer was identified based on its characteristic retention time and absorption spectrum of retinoid standards. Isomerohydrolase activity was calculated from the area of the 11 cROL
peak using Radiomatic 610TR software (Perkin Elmer, Boston, MA) with synthetic 11-cis [3H]-retinol as the standard. To minimize the variation of the transfection efficiency, all in vitro activity assays of the mutants were conducted side-by-side with wt hRPE65, and the catalytic activities were expressed as values relative to that of wt hRPE65 unless specified.

[0141] RESULTS

[0142] Comparison of expression and enzymatic activities of hRPE65 and cRPE65.

[0143] Plasmids expressing red fluorescence protein (rfp; negative control), hRPE65 and cRPE65 were separately transfected into the 293A-LRAT cells and cultured for 48 hr. The expression levels and enzymatic activities of hRPE65 and cRPE65 were verified by Western blot analysis (FIG.3A) and in vitro isomerohydrolase assay. Both hRPE65 and cRPE65, but not negative control protein rfp, converted all-trans retinyl ester into 11 cROL, the product of isomerohydrolase (FIGS.3B-3D). cRPE65 produced substantially higher levels of 11 cROL
than that of wt hRPE65 after the normalization to its RPE65 protein level, indicating a higher enzymatic activity of cRPE65.

[0144] Selection of Mutants.

[0145] Eight residues from various positions in cRPE65 were selected for examining the difference in the isomerohydrolase activity between hRPE65 and cRPE65 (see FIG.1). In order to evaluate the contribution of these residues to isomerohydrolase activity, a series of point mutations of the 8 candidate residues in hRPE65 were generated by site-directed mutagenesis. For in vitro enzyme activity assays, the effect of substitution of Thr at position 39, Asn at position 170, Cys at position 330 and Gln at position 497 in hRPE65 was examined in a first experimental set. Substitutions of Cys at position 106, Lys at position 297, Leu at position 510, and Ser at position 533 were examined in a second experimental set.

[0146] Impacts of site-directed point mutations on protein levels and catalytic activities of hRPE65.

[0147] Plasmids expressing hRPE65 and cRPE65, and the site-directed mutants were separately transfected into 293A-LRAT cells, and the transfected cells were cultured for 48 hr. Protein expression was confirmed by Western blot analysis (relative expression levels of tested RPE65 mutants are shown in Table 2), and the same batches of total cellular proteins were used for the in vitro isomerohydrolase activity assay. For all of the hRPE65 mutants containing single, double and multiple mutations, expression levels of RPE65 were comparable (FIGS.4A-4E), whereas the two single point mutants N170K and K297G
exhibited 1.6-fold and 1.7-fold higher enzymatic activity than that of wt hRPE65, respectively, after normalization by total RPE65 expression levels (FIGS 4F
and 4G).
Furthermore, the tested double, triple, or multiple mutants with N170K (or K297G) in each experimental sets did not further enhance catalytic activity of RPE65. These results suggest that mutations of N170K and K297G in hRPE65 may be important for increasing its enzymatic activity.

[0148] Table 2: Expression levels of generated RPE65 mutants.
Protein expression Mutant Name N=
(% of WT SEM) wt hRPW65 100.0 T39R 119.2 8.3 6 N170K 104.9 3.3 7 C330T 106.3 6.5 6 Q497P 80.6 8.4 6 T39R/N170K 102.3 10.7 5 N170K/C330T 86.2 8.7 5 N170K/Q497P 92.0 4.4 5 T39R/N170K/C330T 92.1 3.0 4 T39R/N170K/Q497P 87.9 2.2 4 T39R/N170K/C330T/Q497P 108.9 5.4 4 C106Y 89.0 5.9 5 K297G 111.3 7.1 7 L510M 171.0 11.7 4 S533A 114.9 9.1 5 , C106Y/K297G 146.3 8.2 3 K297G/L510M 186.1 16.8 3 K297G/S533A 176.3 - 12.2 3 1220M 99.2 1 9.8 4 N170K/1220M 78.7 2.4 3 T39R/N170K/1220M 103.3 0.9 3 T39R/N170K/1220M/Q497P 96.9 2.8 4 N3021 130.4 14.1 3 K297G/N3021 145.2 8.0 4

[0149] Construction of chimeric human RPE65 and its impacts on the protein levels and catalytic activity.

[0150] To further improve the catalytic activity of hRPE65, we constructed chimeric RPE65 by replacing a peptide fragment of hRPE65 with the counterpart of cRPE65 (FIG.5A, fragments F1-F6). At 48 hr post-transfection, the cells were harvested for Western blot analyses and in vitro isomerohydrolase assays. Western blot analysis showed that the chimeric mutants except for a F 1 chimera (replaced fragment 1) displayed higher protein level of RPE65 than that of wt hRPE65 and other chimeric mutants (FIGS.5B-5C).

Interestingly, F 1 and F3 chimeric mutants showed approximately 1.5-2-fold higher catalytic activity than that of wt hRPE65, whereas the F2, F4 and F5 chimeric mutants substantially decreased its catalytic activities of hRPE65. Following the normalization by RPE65 protein levels, the F1 chimeric mutant showed the catalytic activity (106.9% of wt hRPE65) comparable to that of wt hRPE65, suggesting that the enhanced catalytic activity of the F1 chimera was likely due to the increased protein level of the F1 RPE65 chimera (FIGS.5B, 5C). It should be noted that the F3 chimera displayed higher normalized enzymatic activity than wt hRPE65 likely due to N170K mutation (152% of wt hRPE65). This is well-correlated with the result of N170K single mutation. The Fl fragment of cRPE65 comprises residues at positions 2, 3, and 26 which are different from corresponding residues in hRPE65 (S2Y, I3S, and L26V).

[0151] Generation of super-isomerohydrolase (sIMH) by combination of highly active mutants.

[0152] Combinations were made of three highly active mutants found from the series of site-directed point mutants and the chimeric mutant studies. As noted above, two single mutants (N170K and K297G) showed significantly higher catalytic activity than that of wt hRPE65 (FIG.6B), even though their expression levels were similar to that of wt hRPE65 (FIG.6A). As shown in FIG.6, a double mutant (N170K/K297G) of hRPE65 demonstrated enzymatic activity 3.2-fold higher than that of wt hRPE65. In addition, a combination of the Fl chimera (having three substitutions) with the N170K mutations showed even higher (2.2-fold) protein levels and catalytic activity than that of N170K single mutant.
After combining all of these mutations (S2Y/I3S/L26V/N170K/K297G) into a single mutant, (sIMH), the catalytic activity of the mutant was found to be 4.4-fold of that of wt hRPE65, which is comparable to the activity of wt cRPE65 (5.8-fold in the same assay, FIG.6B).

[0153] Alternate mutants.

[0154] As shown in Table 3, certain mutations based on differences between hRPE65 and cRPE65, e.g., at positions 220 and 302, either did not improve activity, diminished activity, or diminished the improvement induced by mutations at positions 170 and 297.

[0155] Table 3: Isomerohydrolase activities of 1220M and N3021 mutant series of hRPE65.
Normalized activity Mutant Name (% of WT SEM) wt hRPE65 100 1220M 62.0 11.0 N170K/1220M 31.1 7.8 T39R/N170K/1220M 109.7 4.2 T39R/ N170K/1220M/Q497P 108.6 10.4 N3021 124.8 13.3 N302/K297G 107.7 6.9

[0156] Two point mutations in particular, N170K and K297G, substantially increased enzymatic activity of hRPE65. The mutant that includes both of these mutations (N170KJK297G) showed a further enhanced catalytic activity (3.2-fold of wt RPE65) of hRPE65. Furthermore, the activity of this double mutant was further enhanced by the _Arg replacement of the Fl fragment from cRPE65 (1met33;containing 3 divergent residues: Y
at position 2, S at position 3, and V at position 26). As a result, the catalytic activity of this super-isomerohydrolase mutant (sIMH; F 1 /N170K/K297G) was approximately 4.4-fold of that of wt hRPE65 under the same experimental condition, and it was contributed by 1.9-fold higher catalytic activity plus 2.3-fold higher protein level. Production of 11 cROL by sIMH
was near the level of wt cRPE65 (5.8-fold of wt hRPE65).

[0157] As further confirmation of enhanced enzymatic activity of sIMH, we evaluated catalytic efficiencies of wt hRPE65 and sIMH (FIG.7). Calculated Km and V.
suggested that higher Vmax of sIMH mainly contributes to its higher enzymatic activity (FIG.7C).

[0158] To understand the potential contribution of the identified key residues to higher catalytic activity of cRPE65, we analyzed the 3D structure of bovine RPE65 (PDB accession:
3FSN) by a SwissPDB Viewer version 4.01 (/www.expasy.org/spdbv/) and displayed the results by a POV-Ray version 3.61 (www.povray.org/). In the 3D model, both Asn170 and Lys297 residues are localized on the surface of RPE65 molecule (FIGS.8A, B) and distant to the co-factor iron in the RPE65 catalytic site (FIG.8C), suggesting that these two residues are unlikely to directly participate in the catalysis of the substrate. Without wishing to be bound by theory, we speculate that these residue substitutions might improve RPE65 membrane association, e.g., for more efficient substrate intake and/or product (11cROL) release.
Alternatively, these two residues might contribute to proper folding of RPE65 to achieve its active conformation upon association with the membrane. Interestingly, the F1 chimera (substituted at positions 2, 3, and 26) also revealed a significant improvement of the catalytic activity of hRPE65 due to increased protein levels of RPE65. Unlike other two residue substitutions, the F1 chimera in which only the Fl fragment is replaced with that of cRPE65 showed catalytic activity similar to that of wt hRPE65 after normalization by RPE65 protein level. The F1 fragment contains 33 amino acids and only 3 residues that differ between hRPE65 and cRPE65. These residues in the F1 fragment might contribute to proper folding to enhance protein stability of the hRPE65 mutant. Another possibility is the contribution of the nucleotide sequence difference. Among the 99 base pairs encoding the 33 amino acids in the F1 fragment, there are 19 different nucleotides between hRPE65 and cRPE65.
These nucleotide substitutions might improve the stability of the hRPE65 mRNA or contribute to more efficient codon usage, leading to higher levels of expression.

[0159] As can be seen from the above, the present work generated, in at least one embodiment, a highly active hRPE65 mutant, (herein designated as super-isomerohydrolase (sIMH)), by substituting wild type 5 residues (at positions 2, 3, 26, 170, and 297) with residues of the corresponding amino acid positions of cRPE65. This sIMH
displayed higher catalytic activity (4.4-fold higher than wt RPE65) after normalization by the total cellular protein levels. In one embodiment, this sIMH mutant can be used in RPE65 gene replacement therapy for more effective treatment of retinal dystrophies in human subjects.
Other substitutions which may be made to form other mutants of the presently disclosed inventive concepts, based on other mammalian RPE65 sequences (including, but not limited to, other mammalian RPE65 sequences described herein), include, but are not limited to, those shown in FIG.9. For example, in one or more non-limiting embodiments, at position 2, Phe or His could be used as the substituted amino acid instead of Tyr, at position 3, Thr or Cys could be used as the substituted amino acid instead of Ser, at position 26, Ala or Ile could be used as the substituted amino acid instead of Val, at position 170, Arg or His could be used as the substituted amino acid instead of Lys, and at position 297, Ala could be used as the substituted amino acid instead of Gly. For example, any of the mutants formed based on SEQ ID NO:1 can also be made in any of the mammalian RPE65 sequences SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID
NO:17.

[0160] In vitro activity assay of indicated mutants were performed as described herein and isomerohydrolase activities were expressed as relative activity following the normalization of RPE65 levels (% of wt hRPE65 activity, mean SEM, n=3).

[0161] While the presently disclosed inventive concepts have been described herein in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the presently disclosed inventive concepts be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications and equivalents are included within the scope of the presently disclosed inventive concepts as defined herein. Thus the examples described above, which include particular embodiments, will serve to illustrate the practice of the presently disclosed inventive concepts, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments of the presently disclosed inventive concepts only and are presented in the cause of providing what is believed to be the most useful and readily understood description of procedures as well as of the principles and conceptual aspects of the inventive concepts. Changes may be made in the formulation of the various compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the presently disclosed inventive concepts. Further, while various embodiments of the presently disclosed inventive concepts have been described in claims herein below, it is not intended that the presently disclosed inventive concepts be limited to these particular claims.

Claims (66)

What is claimed is:

1. A mutant mammalian RPE65 protein or portion thereof, comprising an amino acid sequence having at least 90% identity with at least one of SEQ ID NO:1, SEQ ID
NO:3, SEQ
ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17, and having an amino acid substitution in at least one of positions 170 and 297; said mutant mammalian RPE65 protein or portion thereof having isomerohydrolase activity

2. A mutant mammalian RPE65 protein or portion thereof according to Claim 1, wherein said amino acid substitution in position 170 is Lys, Arg, or His; and said amino acid substitution in position 297 is Gly or Ala.

3. A mutant mammalian RPE65 protein or portion thereof according to Claim 1, further comprising an amino acid substitution in at least one of positions 2, 3, and 26.

4. A mutant mammalian RPE65 protein or portion thereof according to Claim 3, wherein said amino acid substitution in position 2 is Tyr, Phe, or His; said amino acid substitution in position 3 is Ser, Thr, or Cys; and said amino acid substitution in position 26 is Val, Ala, or Ile.

5. A mutant mammalian RPE65 protein or portion thereof according to Claim 1, comprising amino acid substitutions in positions 2, 3, 26, 170, and 297.

6. A mutant mammalian RPE65 protein or portion thereof according to Claim 1, wherein said mutant mammalian RPE65 protein or portion thereof has greater isomerohydrolase activity than non-mutated RPE65 protein.

7. A mutant mammalian RPE65 protein or portion thereof according to Claim 1, wherein said mutant mammalian RPE65 protein or portion thereof has greater isomerohydrolase activity than human RPE65 having the amino acid sequence set forth in SEQ ID
NO:1 or bovine RPE65 having the amino acid sequence set forth in SEQ ID NO:9.

8. A pharmaceutical composition comprising a mutant mammalian RPE65 protein or portion thereof according to Claim 1 disposed in a pharmaceutically-acceptable carrier or vehicle.

9. A nucleic acid encoding a mutant mammalian RPE65 protein or portion thereof according to Claim 1.

10. A nucleic acid according to Claim 9, wherein said amino acid substitution in position 170 is Lys, Arg, or His; and said amino acid substitution in position 297 is Gly or Ala.

11. A nucleic acid according to Claim 10, wherein said mutant mammalian protein or portion thereof further comprises an amino acid substitution in at least one of positions 2, 3, and 26.

12. A nucleic acid according to Claim 11, wherein said amino acid substitution in position 2 is Tyr, Phe, or His; said amino acid substitution in position 3 is Ser, Thr, or Cys;
and said amino acid substitution in position 26 is Val, Ala, or Ile.

13. A nucleic acid according to Claim 9, wherein said mutant mammalian RPE65 protein or portion thereof comprises amino acid substitutions in positions 2, 3, 26, 170, and 297.

14. A nucleic acid according to Claim 9, disposed within a vector.

15. A nucleic acid according to Claim 14, wherein said vector comprises: a promoter or enhancer sequence operatively-linked to said nucleic acid; or a stop codon or a poly-A
sequence located 3 of said nucleic acid.

16. A nucleic acid according to Claim 14, wherein said vector comprises an promoter sequence operatively-linked to said nucleic acid.

17. A nucleic acid according to Claim 14, wherein said vector comprises a CMV
enhancer/chicken p-actin promoter sequence operatively-linked to said nucleic acid.

18. A nucleic acid according to Claim 14, wherein said vector is selected from the group consisting of adenovirus, adeno-associated virus (AAV), nanoparticles, plasmids, and lentivirus.

19. An adeno-associated viral (AAV) vector, comprising a nucleic acid encoding a mutant mammalian RPE65 protein or portion thereof according to any of Claims 1-5.

20. An AAV vector, comprising a nucleic acid according to any of Claims 9-17.

21. An AAV vector according to Claim 19 or 20, where said vector comprises one or more AAV inverted terminal repeat (ITR) sequences.

22. An AAV vector according to Claim 21, wherein said one or more AAV ITR
sequences comprises an ITR sequence of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes, or a mixture of ITR sequences therefrom.

23. An AAV vector according to Claim 21, wherein said AAV vector comprises an AAV
capsid sequence.

24. An AAV vector according to Claim 23, wherein said AAV capsid sequence comprises a VP1, VP2 and/or VP3 capsid sequence having at least 90% identity to the VP1, VP2 and/or VP3 sequences of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8.

25. A cell that produces an AAV vector according to any of Claims 19-24.

26. A gene therapy method of treating a condition related to retinal degeneration in a mammalian subject in need of such treatment, said method comprising:
administering to said subject a therapeutically-effective amount of a vector comprising a nucleic acid which encodes a mutant mammalian RPE65 protein or portion thereof, wherein said mutant mammalian RPE65 protein or portion thereof is expressed in vivo in retinal cells of said subject and has isomerohydrolase activity.

27. A gene therapy method according to Claim 26, wherein said mutant mammalian RPE65 protein mutant or portion thereof encoded by said nucleic acid comprises an amino acid sequence having at least 90% identity with at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID
NO:15, and SEQ ID NO:17, and having an amino acid substitution in at least one of positions 170 and 297.

28. A gene therapy method according to Claim 27, wherein said amino acid substitution in position 170 is Lys, Arg, or His; and said amino acid substitution in position 297 is Gly or Ala.

29. A gene therapy method according to Claim 27, wherein said mutant mammalian RPE65 protein or portion thereof further comprises an amino acid substitution in at least one of positions 2, 3, and 26.

30. A gene therapy method according to Claim 27, wherein said amino acid substitution in position 2 is Tyr, Phe, or His; said amino acid substitution in position 3 is Ser, Thr, or Cys;
and said amino acid substitution in position 26 is Val, Ala, or Ile.

31. A gene therapy method according to Claim 26, wherein said mutant mammalian RPE65 protein or portion thereof has greater isomerohydrolase activity than non-mutated RPE65 protein.

32. A gene therapy method according to Claim 26, wherein said mutant mammalian RPE65 protein or portion thereof has greater isomerohydrolase activity than human RPE65 having the amino acid sequence set forth in SEQ ID NO:1 or bovine RPE65 having the amino acid sequence set forth in SEQ ID NO:9.

33. A gene therapy method according to Claim 26, wherein said vector further comprises a promoter or enhancer sequence operatively-linked to said nucleic acid encoding said mutant mammalian RPE65 protein; or further comprises a stop codon or a poly-A
sequence located 3' of said nucleic acid encoding said mutant mammalian RPE65 protein.

34. A gene therapy method according to Claim 26, wherein said vector is selected from the group consisting of adenovirus, adeno-associated virus (AAV), nanoparticles, plasmids, and lentivirus.

35. A gene therapy method according to Claim 26, wherein said vector comprises an AAV vector which comprises one or more AAV inverted terminal repeat (ITR) sequences.

36. A gene therapy method according to Claim 35, wherein the AAV ITR
sequence comprises an ITR sequence of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes, or a mixture of ITR sequences therefrom.

37. A gene therapy method according to Claim 26, wherein said vector comprises an AAV capsid sequence.

38. A gene therapy method according to Claim 37, wherein said AAV capsid sequence comprises a VP1, VP2 and/or VP3 capsid sequence having at least 90% identity to the VP1, VP2 and/or VP3 sequences of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8.

39. A gene therapy method according to Claim 26, wherein said condition related to retinal degeneration is Leber's Congenital Amaurosis or Retinitis pigmentosa.

40. A method of treating a condition related to retinal degeneration in a subject in need of such treatment, said method comprising:
administering to said subject a therapeutically-effective amount of at least one mutant mammalian RPE65 protein or a portion thereof, thereby mitigating said condition related to retinal degeneration in said subject, said mutant mammalian RPE65 protein or portion thereof having isomerohydrolase activity.

41. A method of treating a condition related to retinal degeneration according to Claim 40, wherein said mutant mammalian RPE65 protein or portion thereof comprises an amino acid sequence having at least 90% identity with at least one of SEQ ID
NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ
ID NO:15, and SEQ ID NO:17; said mutant mammalian RPE65 protein or portion thereof having an amino acid substitution in at least one of positions 170 and 297.

42. A method of treating a condition related to retinal degeneration according to Claim 41, wherein said amino acid substitution in position 170 is Lys, Arg, or His; and said amino acid substitution in position 297 is Gly or Ala.

43. A method of treating a condition related to retinal degeneration according to Claim 41, wherein said mutant mammalian RPE65 protein further comprises an amino acid substitution in at least one of positions 2, 3, and 26.

44. A method of treating a condition related to retinal degeneration according to Claim 41, wherein said amino acid substitution in position 2 is Tyr, Phe, or His; said amino acid substitution in position 3 is Ser, Thr, or Cys; and said amino acid substitution in position 26 is Val, Ala, or Ile.

45. A method of treating a condition related to retinal degeneration according to Claim 40, wherein said condition related to retinal degeneration is Leber's Congenital Amaurosis or Retinitis pigmentosa.

46. A method of treating a condition related to retinal degeneration according to any of Claims 26-45, wherein said subject is human.

47. A method of treating a condition related to retinal degeneration according to any of Claims 26-46, wherein said subject is less than 18 years old, less than 15 years old, less than 12 years old, less than 10 years old, between about 8-10 years old, between about 6-10 years old, between about 4-6 years old, between about 1-3 years old or is less than 1 year old or less.

48. A method of treating a condition related to retinal degeneration according to any of Claims 26-46, wherein said subject has or is at risk of having retinal degeneration or dystrophy caused by RPE65 mutation,

49. A method of treating a condition related to retinal degeneration according to any of Claims 26-39, wherein said vector is administered to one or more eyes of said subject.

50. A method of treating a condition related to retinal degeneration according to any of Claims 26-39, wherein said vector is administered to the subretinal space and/or suprachoroidal space of one or more eyes of said subject.

51. A method of treating a condition related to retinal degeneration according to any of Claims 26-39, wherein said vector comprises an AAV vector and wherein 1×10 8 or more AAV vector genomes are administered to one or more eyes of said subject.

52. A method of treating a condition related to retinal degeneration according to any of Claims 26-39, wherein said vector comprises an AAV vector and wherein about 1×10 8 to about 1×10 14 AAV vector genomes are administered to one or more eyes of said subject.

53. A method of treating a condition related to retinal degeneration according to any of Claims 26-39, wherein said vector comprises an AAV vector and wherein about 1×10 9 to about 1×10 13AAV vector genomes are administered to one or more eyes of said subject.

54. A method of treating a condition related to retinal degeneration according to any of Claims 26-46, wherein said vector or mutant mammalian RPE65 protein or portion thereof is administered in a volume of about 10 microliters (µl) to about 1,000µI.

55. A method of treating a condition related to retinal degeneration according to any of Claims 26-46, wherein said vector or mutant mammalian RPE65 protein or portion thereof is administered in a volume of about 50 µl to about 800 µl.

56. A method of treating a condition related to retinal degeneration according to any of Claims 26-46, wherein said vector or mutant mammalian RPE65 protein or portion thereof is administered in a volume of about 100 µl to about 600 µI.

57. A method of treating a condition related to retinal degeneration according to any of Claims 26-46, wherein said vector or mutant mammalian RPE65 protein or portion thereof is administered in a volume of about 200 µl to about 500 µl.

58. A method of treating a condition related to retinal degeneration according to any of Claims 26-46, wherein said vector or mutant mammalian RPE65 protein or portion thereof is delivered or introduce in cells of the retinal pigment epithelium of one or more eyes of said subject.

59. A mutant mammalian RPE65 protein or portion thereof, comprising an amino acid sequence having at least 90% identity with at least one of SEQ ID NO:1, SEQ ID
NO:3, SEQ
ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17, and having at least one amino acid substitution that results in an about 2-fold increase to an about 6-fold increase in isomerohydrolase activity as compared to wild-type human RPE65.

60. A mutant mammalian RPE65 protein or portion thereof according to Claim 59, wherein said about 2-fold to about 6-fold increase in isomerohydrolase activity is determined by in vitro isomerohydrolase activity assay.

61. A mutant mammalian RPE65 protein or portion thereof according to Claim 59, wherein said at least one amino acid substitution is Lys, Arg, or His at position 170.

62. A mutant mammalian RPE65 protein or portion thereof according to Claim 59, wherein said at least one amino acid substitution is Gly or Ala at position 297.

63. A mutant mammalian RPE65 protein or portion thereof according to Claim 59, wherein said at least one amino acid substitution includes a Lys, Arg, or His substitution at position 170, and a Gly or Ala substitution at position 297.

64. A mutant mammalian RPE65 protein or portion thereof according to Claim 63, wherein said amino acid substitutions result in an about 3.2-fold increase in isomerohydrolase activity compared to wild-type human RPE65.

65. A mutant mammalian RPE65 protein or portion thereof according to Claim 59, wherein said at least one amino acid substitution includes: a Lys, Arg, or His substitution at position 170; a Gly or Ala substitution at position 297; a Tyr, Phe, or His substitution at position 2; a Ser, Thr, or Cys substitution at position 3; and a Val, Ala, or Ile substitution at position 26.

66. A mutant mammalian RPE65 protein or portion thereof according to Claim 65, wherein said amino acid substitutions result in an about 4.4-fold increase in isomerohydrolase activity compared to wild-type human RPE65.